Nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against HIV-1 and HBV. However, both viruses often acquire NRTI resistance, making it crucial to develop more potent agents that offer profound viral suppression. We report here that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel highly potent inhibitor of both HBV (IC50=0.4 nM) and HIV-1 (IC50=0.4 nM). In contrast, the approved anti-HBV NRTI entecavir (ETV) potently inhibits HBV (IC50=0.7 nM) but is much less active against HIV-1 (IC50=1,000 nM). Similarly, the highly potent HIV-1 inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) (IC50=0.3 nM) is less active against HBV (IC50=160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50=7.2 nM) and ETV-resistant HBV (IC50=69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 microM. Once daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (p=0.0005) in human-liver-chimeric/HBVETV-RL180M/S202G/M204V-infected mice, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. At the beginning of the present study, we found that EFdA, a highly potent anti-HIV-1 agent, was much less potent against HBVWT in cell-based assays. On the other hand, ETV proved to be highly active against HBVWT, while it was much less active against HIV-1. Furthermore, AZT, the first known nucleoside analog active against HIV-1 was virtually inert against HBVWT, while LdT that has been used for the treatment of HBV infection was essentially inert against HIV-1. In this regard, HBV-RT, in terms of amino acid sequence homology, is close to HIV-1-RT and murine leukemia virus (MuLV)-RT, but with less than 25% amino acid sequence identity. Nevertheless, the study of sequence alignment performed demonstrated that the functionally important amino acid residues are highly conserved among the RTs of HBV, HIV-1, and MuLV, especially in the catalytic region of those RTs that include the conserved domains A-G. It was thought from the observed differential activity of EFdA, ETV, AZT, and LdT against HIV-1 and HBV that the catalytic regions of HIV-1-RT and HBV-RT should sufficiently and HIV-1-RT and HBV-RT differentially recognize those nucleosides as good or poor substrates. Thus, we hypothesized that we could successfully identify lead compounds active against HBV among modified nucleosides that we examined and found essentially inert against HIV-1 or inappropriate as anti-HIV-1 therapeutics in our initial in vitro screening assays conducted in the 1980s and 1990s. Indeed, both CAdA and CdG were among the modified nucleoside analogs we previously reported to be active against HIV-1. Considering that the multiple antiviral mechanisms involved represent a unique feature of the 4'-modified NRTIs, we hypothesized that CAdA and CdG would also act as efficient HBV DNA chain terminators through the mechanism(s) mentioned above, which should differ from the mechanism(s) through which ETV exerts its activity against HBV and that CAdA and CdG would also be active against HBVETV-RL180M/S202G/M204V. As expected, both CAdA and CdG proved to significantly reduce the production of both HBVWT and HBVETV-RL180M/S202G/M204V in cell-based assays and productively HBV-infected hu-liver-chimeric-uPA+/+/SCID+/+ mice. The IC50 value differencein the HBV signal reduction in the Southern blot between HBVWT and HBVETV-R was 9.7-fold for CAdA treatment and 4.3-fold for CdG treatment. In the mouse model, the difference in the viremia reduction magnitude between HBVWT and HBVETV-R was 30-fold (-2.9 vs -1.4 log10 copies/ml) for CAdA treatment and 30-fold (-2.7 vs -1.2 log10 copies/ml) for CdG treatment. However, it should be noted that the potential efficacy against HBVETV-R of CAdA and CdG is to be determined only in carefully managed clinical trials. In our structural modeling of HIV-1-RT and HBV-RT in complex with ETV-TP, EFdA-TP, or CAdA-TP, there appear to be three major factors contributing to greater potency of ETV against HBV compared to HIV-1: (i) the shallower pocket in HBV-RT compared to HIV-1-RT, (ii) the presence of a phenylalanine (F88) in HBV-RT in place of the slightly polar tyrosine (Y115) in HIV-1-RT that appears to contribute to a stronger hydrophobic interaction with the exocyclic alkene-containing hydrophobic ring of ETV-TP and (iii) favorable interactions of L180 in HBV-RT with ethene-cyclopentyl ring in ETV-TP, which is absent in HIV-1-RT due to the slightly distal location of F160 at equivalent position. Therefore, the stronger hydrophobic interactions of the pseudo-sugar (exocyclic alkene containing cyclopentyl ring) of ETV-TP in HBV-RT compared to those in HIV-1-RT may contribute to the enhanced binding of ETV-TP leading to a greater inhibition of HBV compared to HIV-1, although it is possible that other factors also contribute to the observed potency differences. In the models of EFdA-TP, the 4'-ethynyl binding pocket in HIV-1-RT is deeper compared to that in HBV-R), which may increase the interactions and affinity of EFdA-TP with HIV-1-RT compared to that in HBV-RT. The sugar ring of CAdA-TP has a 4'-cyano instead of the 4'-ethynyl in EFdA. The 4'-cyano group is slightly shorter than the 4'-ethynyl, and both moieties can be accommodated in the deeper hydrophobic pocket of HIV-1-RT, accounting for the similar antiviral potencies of EFdA and CAdA against HIV-1 (IC50 values: 0.4 vs. 0.3 nM). Moreover, the 4'-cyano-containing NRTI-TPs can be accommodated in the shallow pocket of HBV-RT, but the slightly longer 4'-ethynyl may lead to reduced binding or misalignment of EFdA in the same pocket. These subtle differences between lengths of 4'-cyano and 4'-ethynyl may explain to a certain extent the difference in the potency of EFdA-TP compared to that of CAdA in HBV-RT. However, differences in the catabolism of CAdA and EFdA to respective triphosphates in different cell types may also affect the potency of these NRTIs. In conclusion, the data presented in this study suggest that CAdA and CdG represent promising candidates as novel therapeutics for HBVETV-RL180M/S202G/M204V infection and that further structural optimization of 4'-modified nucleosides should help develop more novel NRTIs that have unique and better antiviral profiles against HBV.